Emergency cardiac surgery and heparin resistance in a patient with essential thrombocythemia

Abstract A 66-year-old man with thrombocytosis was brought to our hospital to undergo removal of a left ventricular thrombus. He had developed cerebral infarction 6 days before presenting to the hospital and suffered from right incomplete hemiparalysis. Blood tests on admission revealed his platelet count to be 124.3 × 104/μl. The urgent removal operation was performed under general anesthesia. For carrying out extracorporeal circulation (ECC), approximately three times as much heparin as expected was needed, as well as antithrombin III (AT III) administration. This met the definition of heparin resistance. The thrombus was removed and surgical left ventricular reconstruction was performed. Aspirin and warfarin were initiated on postoperative day 5. A bone marrow biopsy was performed on postoperative day 8, which revealed hypercellular marrow with megakaryocyte proliferation, and the patient was diagnosed as having essential thrombocythemia (ET). Although hydroxycarbamide administration started on postoperative day 10, his platelet count increased to 290.7 × 104/μl on postoperative day 13. The counts descended gradually, and on postoperative day 34, his platelet count reached the normal range and he was discharged from the hospital. In the perioperative period, his new neurologic abnormality did not appear. Addition of heparin, administration of AT III, and coating the cardiopulmonary bypass circuit with heparin or macromolecular polymer prevented clot formation and enabled safe ECC in a patient with ET and a high platelet count

Biochemical Characterization of Medaka (<i>Oryzias latipes</i>) Transglutaminases, OlTGK1 and OlTGK2, as Orthologues of Human Keratinocyte-Type Transglutaminase

<div><p>Calcium-dependent transglutaminases (TGs) are a family of enzymes that catalyze protein cross-linking and/or attachment of primary amines in a variety of organisms. Mammalian TGs are implicated in multiple biological events such as skin formation, blood coagulation, and extracellular matrix stabilization. Medaka (<i>Oryzias latipes</i>) has been used as a model fish to investigate the physiological functions of mammalian proteins. By analysis of the medaka genome, we found seven TGs orthologues, some of which apparently corresponded to the mammalian TG isozymes, TG1, TG2, and Factor XIII. All orthologues had preserved amino acid residues essential for enzymatic activity in their deduced primary structures. In this study, we analyzed biochemical properties of two orthologues (OlTGK1 and OlTGK2) of mammalian epithelium-specific TG (TG1) that are significantly expressed at the transcriptional level. Using purified recombinant proteins for OlTGK1 and OlTGK2, we characterized their catalytic reactions. Furthermore, immunohistochemical analyses of fish sections revealed higher expression in the pancreas (OTGK1), intervertebral disk (OlTGK2) and pharyngeal teeth (OlTGK2) as well as in the skin epidermis.</p></div

The enzymatic activities of OlTGK1 and OlTGK2 evaluated by incorporation of biotin-cadaverine.

<p>The transamidating activities of the purified recombinant OlTGK1 and OlTGK2 proteins were measured by incorporation of biotinylated cadaverine (bio-Cd) into β-casein coated onto wells of microtiter. Incorporation was measured at various concentrations of bio-Cd and with different amounts of the enzymes for 20 min: For OlTGK1 (A), the white, gray, and black circles indicate 2, 5, and 10 ng/μl (final concentrations) of proteins, respectively. For OlTGK2 (C), the white, gray, and black circles indicate 2, 5, and 20 ng/μl of proteins, respectively. Time course reactions (0 to 60 min) with 2 ng/μl of enzyme proteins in the absence (black) and presence (white) of EDTA were shown (B: OlTGK1, D: OlTGK2).</p

Immunohistochemical analysis of OlTGK1 using medaka sections.

<p>Whole body sections of medaka fixed with methanol and acetic acid following by paraffin were analyzed by immunostaining. The serial sections were stained with hematoxylin and eosin (HE) (A) and subjected to immunoreaction with an affinity-purified polyclonal antibody against OlTGK1 (B) as well as a rabbit immunoglobulin solution at a similar concentration (C). The whole body image (B) around the stained area was enlarged; pancreas (D), Brockmann body (E), and salivary gland (F). The scale bars indicate 2 mm (A) and 100 μm (D-F).</p

Immunostaining for OlTGK1 and OlTGK2 in the skin epidermis.

<p>Serial frozen tissue sections prepared and without fixation were subjected to immunohistochemical analysis using polyclonal antibodies as described in the legend to Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144194#pone.0144194.g004" target="_blank">4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144194#pone.0144194.g005" target="_blank">5</a>. The analysis focused on areas of the epidermis: OlTGK1 (A), OlTGK2 (B), and negative controls (C, D) are shown. Scale bar indicates 100 μm.</p

Purification of OlTGK1 and OlTGK2 recombinant proteins produced in bacteria.

<p>The soluble fraction containing recombinant OlTGK1 (A) and OlTGK2 (B) proteins were purified using metal ion affinity-gel and size exclusion chromatography. Each sample was subjected to 7.5% SDS-PAGE following Coomassie Brilliant Blue staining: lane 1; applied samples for affinity chromatography, lane 2; flow-through fractions, lane 3; washed fractions, lane 4; eluted fractions, lane 5; the peak fractions from the size separation performed using Superdex-200 increase. M: molecular mass marker.</p

Sequence alignment of human TGs and medaka orthologues and phylogenic tree based on primary structure.

<p>Sequence alignment around the catalytic triad including Cys (the active-site), His, and Asp was shown with respect to the major human TGs and the medaka orthologues: Human TG1 (NP_000350), TG2 (NP_004604), FXIII (NP_000120), as well as medaka orthologues. The catalytic triad, containing the active site Cys and the other two amino acid residues (Asp, His) is indicated by arrows. The possible residues coordinating calcium ion are indicated as circles. The numbers of the amino acid residues are described with the sequences. (A). The phylogenic tree was constructed (1000 boostrap trials, Neighbor-Joining Method plot) based on the deduced amino acid sequences for human FXIII, TG1, TG2, TG3 (NP_003236), TG4 (NP_003232), TG5 (NP_963925), TG6 (NP_945345), TG7 (NP_443187), and their medaka orthologues. Each cDNA sequence of medaka orthologues has been deposited to DDBJ (DNA Data Bank Japan) as following accession numbers: OlTGB (LC068825), OlTGT (LC068826), OlTGK1 (LC068829), OlTGK2 (LC068830), OlTGK3 (LC068831), OlTGF (LC068827), and OlTGO (LC068828) (B).</p